Impact of the preparation technique on DNA content measured by image analysis in early stage human testis cancer

Current clinical staging which includes serum tumour markers and imaging techniques fails to identify 30-40% of clinical stage I nonseminomatous germ cell testicular tumour (NSGCT) patients who have occult metastatic disease at time of orchiectomy and who will, therefore, develop clinically evident metastases, usually within two years of follow-up. Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumour which might be useful as predictors for occult metastatic disease. Some investigators have described that DNA content measured by image cytometry is of prognostic value in early stage NSGCT to detect patients at risk for occult metastatic disease. However, optimal preparatory techniques are mandatory in establishing new tumour biological markers in order to obtain reliable and reproducible results. This study has analyzed the impact of the sedimentation technique in comparison to the cytocentrifugation technique on DNA measurement in early stage NSGCT obtained by image cytometry. Different tissue blocks of formalin fixed, paraffin embedded primary testicular tumours (NSGCT) of 50 clinical stage I patients, who underwent retroperitoneal lymph node dissection between 1985 and 1989, were analyzed. Thirty (60%) patients had histologically proven lymph node involvement (pathological stage B), whereas 20 (40%) patients (pathological stage A) had neither lymph node metastases nor tumour recurrence during follow-up. The samples were prepared according to a modified Hedley technique: Individual tissue digestion times were monitored closely to avoid overdigestion. The times varied from 30 to 60 min depending on the constituents of the tissue section. Prolonged digestion times did not correlate with poor quality of the preparations and brief digestion times did not always yield optimal specimens. The impact of two different techniques (cytocentrifugation and gravity sedimentation) on the Feulgen staining results were compared. Cytocentrifuged samples consistently provided larger and paler nuclei with less well defined borders compared to slides from the same cell suspension processed by the sedimentation technique. Nuclei from pathologic stage II samples were more vulnerable to cytocentrifuge alteration than those of stage I. According to the results obtained in this study, the sedimentation slide preparation technique should be preferred for DNA ICM in NSGCT, and possibly in other human malignancies as well.